Protein Mobility Inside Pyruvate Dehydrogenase Complexes as Reflected by Laser‐Pulse Fluorometry

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Abstract
The fluorescence decay curves of the flavine in all pyruvate dehydrogenase complexes studied are consistent with a 2-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In 3 of the 4 complexes investigated, about equal weights for the amplitudes of the 2 lifetimes are found. In the 3-component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme-free complex is compared with complex to which Mg2+ and thiamine diphosphate are added. The correlation time calculated from the polarized decay of the flavine fluorescence at 11.degree. C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes, respectively. This correlation time is of the same order as the rotational correlation time of free lipoamide dehydrogenase itself, but much shorter than would be expected from the MW of the complexes. Models explaining the 2 lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.